Project Summary Lung infections and inflammation are major causes of morbidity and mortality worldwide. Uncontrolled lung infection and its associated inflammatory injuries lead to acute respiratory distress syndrome (ARDS), for which no specific treatment is available. An ideal therapeutic would be one that can control pathogen burden and suppress the inflammatory response. Using computational, immunochemical, and functional screening approaches, a Toll-like receptor (TLR4)-interacting surfactant protein-A derived peptide called SPA4 (amino acids: GDFRYSDGTPVNYTNWYRGE) has been identified. The SPA4 peptide binds to the TLR4, suppresses the TLR4-induced inflammation, and yet maintains the TLR4-induced pathogen-recognition, uptake and intracellular processing. The project hypothesis is that the SPA4 peptide will reduce the pathogen burden and inflammatory response during lung infection and inflammation by interacting with and altering the assembly of the TLR4 complex. The specific aims are to: (1) define the molecular basis of the pro-phagocytic and anti-inflammatory effects of the SPA4 peptide, (2) evaluate the biological effects of SPA4 peptide in mouse models of lung infection and inflammation, and (3) analyze the activity of SPA4 peptide on TLR4-signaling and immune function. We will determine the amino acids and motif of SPA4 peptide that are critical for binding to TLR4 and activity against infectious stimuli. We will investigate the TLR4-assembly with its adaptor molecules in human cells and models using structural biology, computational, molecular, biochemical, and cellular approaches. The bacterial uptake and clearance and inflammatory parameters will be simultaneously assessed in primary human cells. Mouse models of Pseudomonas aeruginosa- and lipopolysaccharide- induced lung infection and inflammation will be used to test the biological relevance of SPA4 peptide. We will investigate the SPA4 peptide?s toxicity and immunogenicity, and efficacy of SPA4 peptide upon repeat administration, in immunosuppressed mice, in secondary challenge model, and when administered in combination with conventional antibiotic, surfactant, or anti-inflammatory agent. Bacterial burden, inflammatory parameters, tissue pathology, lung function, symptoms and survival will be assessed. The binding, distribution, pharmacokinetics, and activity of SPA4 peptide will be evaluated for modulation of TLR4-dependent mechanisms at cellular and tissue levels in lungs of wild type and genetic mouse models of TLR4 and its adaptors, using in vivo imaging, immunohistochemistry, flow cytometry, and confocal microscopy. It is expected that the end-point determinants of the proposed studies and analyses will eventually help establish the mechanism of action of the SPA4 peptide. This project uses the unique concept of developing a novel peptide-based immunotherapeutic. We anticipate that the results of this study will facilitate the development of SPA4 peptide as an immunotherapeutic to help reduce pathogen burden and inflammation, improve the clinical condition and survival of patients suffering from lung infection and inflammatory injuries, and control ARDS.